Surfactant p20

Purified mAb was immobilized on a protein G chip through a 30 s injection of 20 nM mAb. CyRPA was diluted in PBS + P20 running buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 0.005% surfactant P20 (GE Healthcare)) to yield a top concentration of 125–500 nM (depending on antibody affinity and required concentration range). Samples were injected for 60 s at 30 μL/min before dissociation for 600 s. The chip was then regenerated with a 45 s injection of 10 mM glycine pH 1.5. Antibody kinetics were determined through a two-fold, six-step dilution curve. Data were analyzed using the Biacore X100 Evaluation sample. A global Langmuir 1:1 interaction model was used to determine antibody kinetics.
For kinetic analyses in the presence of calcium or magnesium, the same procedure was used starting from a top concentration of 100 nM, using TBS + P20 (150 mM NaCl, 20 mM Tris pH 7.4, 0.005% surfactant P20) with or without 1 mM CaCl₂ or MgCl₂. TBS was used in place of PBS due to the presence of visible precipitate for PBS with 1 mM CaCl₂.

Antigenicity of the influenza formulations was quantified by a SPR method modified from Estmer Nilsson et al[15] (link). Samples were analyzed on a Sensor Chip CM5 with a Biacore T200 biosensor system (GE Healthcare). HBS-EP+ (GE Healthcare) was used as analysis buffer. Recombinant HA protein from A/PR/8/34 (Protein Sciences) was immobilized to 7000–10000 response units using an Amine coupling kit (GE Healthcare) with ∼65 μL rHA (10 μg/mL) in 10 mM phosphate buffer, 0.05% Surfactant P20 (GE Healthcare), pH 6.0. Dilutions series of the vaccine samples were made, and anti-influenza A/PR/8/34 sheep serum (1∶150, NIBSC) was added to each dilution. The sample-serum mixture was subsequently injected during 400 seconds during which sensorgrams were acquired. In between each sample the sensor chip surface was regenerated using 50 mM HCl, 0.05% Surfactant P20. Acquired sensorgrams were analyzed using Biacore T200 evaluation software (GE Healthcare). Antigenicity was calculated relative to a known concentration of rHA A/PR/8/34.

4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES, ≥99.5%), ethylenediaminetetraacetic acid (EDTA, ≥99.4%) concentrated saline sodium phosphate-EDTA (20× SSPE, pH 7.4) and DNA from herring sperm (D7290) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium chloride (NaCl, 99.5%) was supplied by PanReac AppliChem (Barcelona, Spain). Biotin CAPture Reagent, regeneration stock 1 and 2 (Biotin CAPture kit reagents) and surfactant P20 were obtained from GE Healthcare (Chicago, IL, USA). The buffers used were: (i) running buffer: HBS-EP (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% (v/v) surfactant P20); and (ii) hybridization buffer: SSPE (2× SSPE, pH 7.4). The DNA oligonucleotides (probes and target sequences) used in this work were purchased from NZYTech (Lisbon, Portugal), and the primers were synthesized by STAB Vida (Caparica, Portugal) as desalted products. Their sequences are listed in S1 Table. All stock solutions were prepared in Milli-Q water (specific resistivity 18.2 MΩ cm) and stored at -20°C.

Surface plasmon resonance (SPR) experiments were performed using a BIAcore X100 instrument (GE Healthcare, Piscataway, NJ) as previously described.33 (link) Biotinylation of atypical LPS types B and B2 was carried out with EZ-Link Sulfo-NHS-LC-Biotin (Pierce Biotechnology). Biotinylated B and B2 LPS were separately immobilized onto streptavidin sensor chips (GE Healthcare). For each sensor chip, a flow cell was left unmodified for reference subtraction. The analysis was conducted by using 1× HBS-EP+ (10 mM N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid], 150 mM NaCl, 3 mM ethylenediaminetetraacetic acid, and 0.05% v/v Surfactant P20; GE Healthcare) as a running buffer and diluent. To evaluate binding affinity, at least six different concentrations of mAbs were used. For each running cycle, the mAb was injected over the surface of a sensor chip at a flow rate of 30 μL/minute for 180 seconds. After this time, the mAb was allowed to passively dissociate for 300 seconds. The sensor surface was regenerated between runs with a 30-second pulse of 10 mM NaOH to ensure the removal of residually bound mAb. The steady-state affinity (K_D) was determined using a steady-state model in BIAevaluation software version 2.0.1 (GE Healthcare). Accuracy of the model fitting was described by χ² parameter calculated by the BIAevaluation software.

Purified SARS-CoV S WT or SARS-CoV S 2P ectodomain was captured on an NTA sensor chip via an 8X HisTag to a level of ∼330 response units (RU) per cycle using a Biacore X100 (GE Healthcare). The NTA sensorchip was regenerated between cycles using 350 mM EDTA and 100 mM NaOH followed by 0.5 mM NiCl₂. A sample containing 10 mM HEPES pH 8.0, 150 mM sodium chloride and 0.05% Surfactant P20 (GE Healthcare) (HBS-P+) was injected over the SARS-CoV S and reference flow cells, followed by injections of purified ACE2 serially diluted 2-fold from 100 nM to 6.25 nM in HBS-P+, with a duplication of the 25 nM concentration. The data were double-reference subtracted and fit to a 1:1 binding model using BIAevaluation analysis software.

To determine the dissociation constants of the designs to the mota or 101F antibodies, surface plasmon resonance was used. Experiments were performed on a Biacore 8K at room temperature with HBS-EP+ running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3mM EDTA, 0.005% v/v Surfactant P20) (GE Healthcare). Approximately 1200 response units of mota or 101F antibody were immobilized via amine coupling on the methyl-carboxyl dextran surface of a CM5 chip (GE Healthcare). Varying protein concentrations were injected over the surface at a flow rate of 30 μl/min with a contact time of 120 sec and a following dissociation period of 400 sec. Following each injection cycle, ligand regeneration was performed using 3 M MgCl₂ (GE Healthcare). Data analysis was performed using 1:1 Langmuir binding kinetic fits within the Biacore evaluation software (GE Healthcare).

Sensor Chip Carboxy Methyl 5 (CM5), N-ethyl-N’-(dimethyl aminopropyl)-carbodiimide (EDC), N-hydroxysuccinimide (NHS), ethanolamine-HCl, phosphate buffer saline (PBS), surfactant P20, glycine HCl (hydrochloric acid) sampling vials and caps were obtained from GE Healthcare Life Sciences, Uppsala, Sweden. The molecules like galanthamine hydrobromide, lycorine hydrochloride, crocin, acetylthiocholine iodide (ATCI), 5,5^ꞌ-dithiobis-(2-nitrobenzoic acid) (DTNB), carbachol, rutin hydrate, pyrogallol, quercetin, catechin hydrate and acetylcholinesterase from Electrophorus electricus (Electric eel) were purchased from Sigma (St. Louis, MO, USA) whereas, tannic acid, caffeine was obtained from Himedia, Mumbai, India. Galanthamine drug (Galamer 4) (Sun Pharmaceutical Industries Ltd, India) was purchased from a pharmacy shop from local market of Kolhapur, India. All chemicals in this study were of the highest purity and molecular purity grade. Milli Q (Millipore, Sigma, USA) water was used for preparing buffers and reagents.